Laping N J, Olson B A, DeWolf R E, Albrightson C R, Fredrickson T, King C, Chirivella M, Ziyadeh F N, Nambi P
Department of Renal Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA.
Biochem Pharmacol. 1998 Jan 15;55(2):227-34. doi: 10.1016/s0006-2952(97)00461-9.
Hepatocyte growth factor (HGF) induces mitogenesis, chemotaxis, and tubule formation in renal epithelial cells. This study examined the effects of wortmannin and protein kinase C (PKC) inhibitors on HGF-mediated changes in metabolic activity in glomerular mesangial cells and renal epithelial carcinoma A498 cells. The extracellular acidification rate of transformed mouse glomerular mesangial cells and A498 cells was measured as an index of metabolic activity with a microphysiometer. HGF increased the acidification rate of mesangial cells and A498 cells in a concentration-dependent fashion that was inhibited completely by the tyrosine kinase inhibitor tyrophostin-23 (100 microM). The PKC inhibitors RO-32-0432 and SKF-57048 also inhibited HGF-induced acidification. The IC50 values for SKF-57048 were 59 +/- 2 and 20 +/- 10 nM in mesangial cells and A498 cells, respectively (P < 0.05). 12-O-Tetradecanoylphorbol 13-acetate (TPA), a phorbol ester that activates PKC, increased acidification in mesangial and epithelial cells similar to HGF. Wortmannin, an inhibitor of phosphatidylinositol (PI) 3-kinase (IC50 value 1-10 nM), inhibited HGF-induced acidification with an IC50 of 93 +/- 31 and 9 +/- 1 nM in mesangial and A498 cells, respectively (P < 0.05). In contrast, there was no significant difference in the IC50 value of wortmannin for epidermal growth factor (EGF)-induced acidification between mesangial and A498 cells (23 +/- 9 vs 14 +/- 1 nM, respectively). Because the IC50 value for wortmannin in inhibiting HGF but not EGF-induced acidification was an order of magnitude higher in mesangial cells than in epithelial A498 cells, a wortmannin-sensitive PI 3-kinase pathway may not be involved in HGF-mediated acidification in mesangial cells.
肝细胞生长因子（HGF）可诱导肾上皮细胞发生有丝分裂、趋化作用和小管形成。本研究检测了渥曼青霉素和蛋白激酶C（PKC）抑制剂对HGF介导的肾小球系膜细胞和肾上皮癌细胞A498细胞代谢活性变化的影响。使用微生理仪测量转化的小鼠肾小球系膜细胞和A498细胞的细胞外酸化率，以此作为代谢活性指标。HGF以浓度依赖性方式增加系膜细胞和A498细胞的酸化率，酪氨酸激酶抑制剂 tyrphostin-23（100 microM）可完全抑制该作用。PKC抑制剂RO-32-0432和SKF-57048也可抑制HGF诱导的酸化。SKF-57048在系膜细胞和A498细胞中的IC50值分别为59±2和20±10 nM（P<0.05）。12-O-十四烷酰佛波醇-13-乙酸酯（TPA）是一种激活PKC的佛波酯，其增加系膜细胞和上皮细胞酸化的作用与HGF相似。渥曼青霉素是磷脂酰肌醇（PI）3激酶的抑制剂（IC50值为1-10 nM），在系膜细胞和A498细胞中，其抑制HGF诱导酸化的IC50分别为93±31和9±1 nM（P<0.05）。相比之下，渥曼青霉素对表皮生长因子（EGF）诱导的系膜细胞和A498细胞酸化的IC50值无显著差异（分别为23±9和14±1 nM）。由于渥曼青霉素抑制HGF而非EGF诱导酸化的IC50值在系膜细胞中比在A498上皮细胞中高一个数量级，因此对渥曼青霉素敏感的PI 3激酶途径可能不参与HGF介导的系膜细胞酸化过程。
